柑橘黄龙病病原菌nrdB基因的原位杂交分析

doi:10.16420/j.issn.0513-353x.2022-1095

摘要/Abstract

摘要：

柑橘植株是否感染黄龙病，可通过qPCR检测其病原韧皮部杆菌（Candidatus Liberibacter Asiaticus，CLas）来判断，但是不同组织样本影响检测结果，因此有必要研究韧皮部杆菌在不同组织中的定位。以柑橘黄龙病韧皮部杆菌的核糖核苷酸还原酶（Ribonucleotide Reductase，RNR）β亚基基因序列为依据，设计和制备123 bp的RNA探针，经原位杂交试验，在显微镜下能清晰地观察到阳性植株筛管和伴胞中的可视化蓝色杂交信号，导管呈环纹结构。CLas在不同器官中的分布存在显著差异，与新生组织根尖和茎尖相比，RNR基因在枝皮、叶柄、叶脉的韧皮部中的定位更准确。原位杂交的可视化结果与qPCR检测结果一致。

关键词: 柑橘, 韧皮部杆菌, RNA原位杂交, 核糖核苷酸还原酶β亚基基因

Abstract:

Candidatus Liberibacter asiaticus（CLas），can be determined by qPCR detection. However，the selection of different tissue samples will affect the results of qPCR detection. Therefore，it is necessary to study the location of CLas in citrus tissues. Based on the specific gene sequence of ribonucleotide reductase（RNR）β subunit of CLas of citrus Huanglongbing disease（HLB），123 bp antisense RNA probe and sense RNA probe was designed and prepared. In situ hybridization analysis was performed on the pathogen gene of CLas in shoot tip，leaf vein，petiole，clade and root tip，meanwhile，the pathogens were detected by qPCR. The results showed that the visual blue hybridization signals in sieve tube and companion cells of phloem in CLas-infected plants could be clearly observed under the microscope and the distribution of pathogens in the different organs was significantly different，compared with root tip and shoot-tip，RNR gene distribution in phloem of bark，petiole and vein was more accurate locating. The HLB-infected condition detected by qPCR and the visualization results of in situ hybridization shared the consistency in citrus nursery plants.

Key words: citrus, Candidatus Liberibacter asiaticus, RNA in situ hybridization, ribonucleotide reductase β subunit gene

张少然, 宗琪, 刘阳, 姜玲. 柑橘黄龙病病原菌nrdB基因的原位杂交分析[J]. 园艺学报, 2023, 50(12): 2689-2700.

ZHANG Shaoran, ZONG Qi, LIU Yang, JIANG Ling. In Situ Hybridization Analysis of Distinctive Ribonucleotide Reductase β Subunit Gene from Candidatus Liberibacter Asiaticus of Citrus[J]. Acta Horticulturae Sinica, 2023, 50(12): 2689-2700.

图/表 10

表1 FAA固定样品的脱水程序

Table 1 Dehydration procedures of the samples for FAA fixed mL

程序 Procedure	蒸馏水 Distilled water	95%乙醇 95%Ethanol	叔丁醇 Tert-butylalcohol
第1次（0 h）The first time	4.0	5.0	1.0
第2次（1~ 4 h）The second time	3.0	5.0	2.0
第3次（1~ 4 h）The third time	1.5	5.0	3.5
第4次（1~ 4 h）The fourth time	0	5.0	5.0
第5次（1~ 4 h）The fifth time	0	2.5	7.5
第6次（0.5 ~ 2 h）The sixth time	0	0	10.0
第7次（0.5 ~ 2 h）The seventh time	0	0	10.0

表2 样品的浸蜡程序

Table 2 Dipping Procedure of wax for samples

程序 Procedure	各成分占比例The proportion of each component		石蜡温度/℃ Temperature of paraffin
程序 Procedure	叔丁醇Tert-butylalcohol	石蜡Paraffin	石蜡温度/℃ Temperature of paraffin
第1次The first time	2/3	1/3	48
第2次The second time	1/2	1/2	48
第3次The third time	1/3	2/3	48
第4次The fourth time	0	1/1	48
第5次The fifth time	0	1/1	54
第6次The sixth time	0	1/1	58

图1 探针制备用到的限制性酶切位点的序列信息括号中的数值指碱基的位置。

Fig. 1 Sequence information of restriction sites used in probe preparation The value in parentheses refers to the position of the base.

表3 qPCR检测黄龙病所用的引物

Table 3 Primers used in qPCR detection of Huanglongbing disease

引物 Primer	引物序列（5′-3′） Primer sequences	片段长度/bp Fragment length	GenBank 编号 GenBank Location accessory	参考文献 Reference
COX+	gtatgccacgtcgcattccaga	68	CX297817 72-93	Li et al.，2006
COX-	gccaaaactgctaagggcattc	68	CX297817 118-139	Li et al.，2006
RNRf	catgctccatgaagctaccc	80	CP010804.1 5434-5455	Zheng et al.，2016
RNRr	ggagcatttaaccccacgaa	80	CP010804.1 5493-5514	Zheng et al.，2016

图2 韧皮部杆菌psy62中5个核糖核酸还原酶β-亚基基因（nrdB）相关序列比对

Fig. 2 Alignment of five ribonucleotide reductase β subunit gene（nrdB）related sequences in Candidatus Liberibacter asiaticus strains psy62

图3 探针制备时测序结果（a）及RNA的反义链凝胶电泳图（b） M：RNA 标样;1：RNR特异片段的反义链探针;2：正义链探针。

Fig. 3 Sequencing results during probe preparation（a）and antisense strand gel electrophoresis of RNA（b） M：RNA marker;1：Anti-sense RNA probe of special fragment of ribonucleotide reductase（RNR）β subunit;2：Sense RNA probe.

图4 ‘爱莎'杂柑叶柄切片上黄龙病菌RNR基因在韧皮部中的表达特征 A：采用RNA反义链探针杂交，韧皮部筛管呈现蓝色信号（150×）;B：以RNA正义链探针杂交作对照，没有产生蓝色杂交信号（150×）;C：杂交信号呈现出大小不等的蓝色颗粒状（500×）;D：显微镜下筛管和伴胞中蓝色杂交信号清晰可见（目镜10×，物镜20×）。s：筛管;c：伴胞;ph：韧皮部;xy：木质部。

Fig. 4 Expression characteristics of RNR gene in phloem of citrus petiole A：On petiole sections of‘Aisha'hybrid（Citrus reticulata‘Ehime 38'× C. reticulata Blanco）infected by Huanglongbing disease，hybridization with RNA antisense strand probe it was showed blue signal in sieve tube of phloem（150×）;B：In the control，no blue hybridization signal was generated in phloem with RNA sense probe hybridization（150×）;C：At a magnification of 500 times，the hybridization signal presented blue granules of varying sizes;D：Under an Olympus BX63 microscope，the blue hybridization signal was clearly visible in the sieve tube and companion cell（eyepiece 10×，objective 20×）. s：Sieve tube;c：Companion cell;ph：Phloem;xy：Xylem.

图5 ‘爱莎'嫩枝皮纵切片（A）、叶柄横切片（B）、叶脉纵切片（C）、根尖纵切片（D）RNA反义链探针的原位杂交信号 A、D采用VHX-6000超景深三维显微系统观察，B、C采用Olympus CX23显微镜观察。ph：韧皮部，xy：木质部。

Fig. 5 In situ hybridization signals of RNA antisense strand probes in longitudinal branch peel section（A），petiole crosscut slice（B），vein longitudinal section（C），and root tip longitudinal section（D）of‘Aisha' A and D were observed by VHX-6000 ultra-depth of field three-dimensional microscopic system，B and C were observed by Olympus CX23 microscope. Ph：Phloem;xy：Xylem.

图6 ‘南丰蜜橘SS-28'嫩枝皮纵切片（A）、叶脉纵切片（B）、叶柄纵切片（C）、茎尖纵切片（D）RNA反义链探针的原位杂交信号 A、B采用Olympus CX23显微镜观察，C、D采用VHX-6000超景深三维显微系统观察。ph：韧皮部;xy：木质部;lp：叶原基;m：生长点。

Fig. 6 In situ hybridization signals of RNA antisense strand probes in longitudinal section（A），longitudinal section（B），longitudinal section of petiole（C）and longitudinal section of stem tip（D）of‘Nanfeng Mandarin SS-28' G and H were observed by VHX-6000 ultra-depth of field three-dimensional microscopic system，E and F were observed by Olympus CX23 microscope. ph：Phloem;xy：Xylem;lp：Leaf primordium;m：Meristem.

表4 柑橘不同器官中韧皮部杆菌中RNR基因的相对表达量分析

Table 4 Relative expression levels of RNR genes in phloem bacilli in different organs of citrus

品种 Cultivar	样品 Sample	CT_RNR	CT_COX	∆∆CT	∆∆CT平均值 ∆∆CT mean	2^-∆∆CT	黄龙病 HLB
对照Control	阴性对照Negative control	28.583 ± 0.180	14.517 ± 0.157	0	0	1	-
对照Control	阳性对照Positive control	31.964 ± 0.944	23.549 ± 0.270	-5.421	-5.651	50.25	+
爱莎Aisha	枝皮Bark	29.362 ± 1.713	16.886 ± 0.435	-2.963	-1.59	3.01	+
	叶脉Veins	29.011 ± 2.202	17.054 ± 0.471	1.710	-2.108	4.31	+
	根Root	30.834 ± 1.418	15.240 ± 0.294	3.593	1.529	0.35	-
	茎尖Shoot tip	33.835 ± 1.728	18.306 ± 0.110	3.764	1.463	0.36	-
	叶柄Petiole	28.047 ± 3.002	14.748 ± 0.110	-4.645	-0.767	1.70	+
南丰蜜橘SS-28 Nanfeng Mandarin SS-28	枝皮Bark	28.845 ± 1.115	16.691 ± 0.218	-3.006	-1.912	3.76	+
	叶脉Veins	31.175 ± 1.616	17.277 ± 0.044	-0.028	-0.167	1.12	+
	根Root	34.692 ± 0.280	16.536 ± 0.438	3.304	4.091	0.06	-
	茎尖Shoot tip	33.298 ± 1.978	16.476 ± 0.072	4.108	2.757	0.15	-
	叶柄Petiole	34.349 ± 1.144	16.409 ± 0.688	4.703	3.875	0.07	-

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